Reactions between biological molecules exhibit an extremely high degree of specificity. It is this specificity that provides a living cell with the ability to carry out thousands of chemical reactions simultaneously in the same "vessel". In general, this specificity arises from the "fit" between two molecules having very complex surface topologies. For example, an antibody binds a molecule displaying an antigen on its surface because the antibody contains a pocket whose shape is the complement of a protruding area on the antigen. This type of specific binding between two molecules forms the basis of numerous biological assays.
For example, the binding between an antibody and molecules displaying a particular antigenic group on their surface may be used as the basis for detecting the presence of the antibody, molecules carrying the antigenic group, or the antigenic group itself. This type of assay forms the basis of numerous medical diagnostic tests. All of these tests depend on detecting and measuring the binding of an antibody molecule that is specific for a particular antigenic group to a molecule carrying the group in question. In general, one of the two molecular species is immobilized on a support surface where it acts as a "glue" for binding the other species. In one class of assays, in which either the antibody or the molecule carrying the antigenic group is to be assayed, one of the two species is covalently immobilized to the support and the other is free in solution. The immobilized species is exposed to the solution that may contain the soluble species and the amount of material bound to the immobilized species after the exposure is measured. In a second class of assays in which the antigenic group itself is to be assayed, one of the two species is covalently immobilized to the support and the other is electrostatically bound to the covalently immobilized species. A solution containing a small molecule having the antigenic group thereon will interfere with the electrostatic binding. This leads to the release of the electrostatically bound species. These assays detect the degree of release of the electrostatically bound species.
Antibodies and antigen carrying molecules are but one of a number of classes of biological molecules whose binding can form the basis of an analytic procedure. For example, nucleic acids are linear polymers in which the linked monomers are chosen from a class of 4 possible sub-units. In addition to being capable of being linked together to form the polymers in question, each unit has a complementary sub-unit to which it can bind electrostatically. For example, in the case of DNA, the polymers are constructed from four bases that are usually denoted by A, T ,G, and C. The bases A and T are complementary to one another, and the bases G and C are complementary to one another. Consider two polymers that are aligned with one another. If the sequences in the polymers are such that an A in one chain is always matched to a T in the other chain and a C in one chain is always matched to a G in the other chain, then the two chains will be bound together by the electrostatic forces. Hence, an immobilized chain can be used to bind the complementary chain. This observation forms the basis of tests that detect the presence of DNA or RNA that is complementary to a known DNA or RNA chain. Such detection forms the basis of a number of medical and/or diagnostic tests.
While this type of specific electrostatic binding provides a high degree of specificity, the amount of material that is bound is usually quite small. This makes the detection of the binding between the reactants difficult. Numerous inventions have been directed to overcoming this problem.
The most sensitive techniques are based on the use of radioisotopes. Unfortunately, any reaction requiring the use of such isotopes presents safety issues which essentially eliminate the assay from being used outside the research laboratory. There are a number of tests that use various labeled chemicals to detect the amount of target material bound to a substrate. Additionally, a sequence of reactants can be bound to the target reactant for purposes of increasing the sensitivity. These tests require the operator to perform a number of processing steps after the reactants have been given a chance to bind. These additional steps detract from the tests in question. In addition, expensive additional radioactive and/or otherwise labeled chemicals are required.
Techniques that measure the binding of the reactants without the need to add radioactive or otherwise labeled chemicals are also known to the art. In one such technique, the reactant that is immobilized on the surface is patterned to form an optical grating. This is accomplished by immobilizing a layer of the reactant to the surface and then inactivating regular strips of the immobilized reactant. The remaining periodic strips will bind the complementary reactant. When the surface is illuminated with a coherent light source such as a laser, the remaining periodic strips act as an optical grating. The presence of the bound material increases the thickness of the strips and may be detected by shifts in the diffraction pattern generated by the grating. While this technique does not require the complex chemistry of the prior art techniques described above, it has two problems that limit its usefulness. First, the system requires optical components such as lenses to direct the selected diffraction order onto an optical detector. Second, the signal energy is distributed among the various diffraction orders in a one-dimensional fashion; hence, the amount of light available for detection is small.